Nanoparticle research is an area of intensive and extensive research, largely due to the changes in physical properties of materials as they approach the 10 nm size range, where among other factors, quantum confinement in semiconductor particles and plasmon resonance can be achieved (Hewakuruppu, et al, 2013). They have a plethora of applications including acting as a semiconductor or sensor, or in biomedical applications as therapeutic agents and vaccines.
Nanoparticles are broadly defined as objects which behave as a single, wholly contained unit with dimensions generally in the range of 1 to 100 nanometers. Their composition is varied and includes a full spectrum of pure or composite materials which can range from metals, such as gold or silver, to biological based particles, such as viruses or engineered virus-like particles (VLP). Typically, virus particles, due to their complexity and requisite storage of genetic information, usually fall toward the upper end of the nanoparticle definition. For example, parvovirus, among the smallest viruses, are particles of approximately 260 Å or 26 nanometers in diameter.
With regard to the prior art, relevant to those biological-based self-assembling nanoparticles (VLP) of non-viral origin, it has previously been disclosed that ferritin, as one such non-viral particle, is the most appropriate and current example of prior art for comparison to the present invention as detailed herein.
The ferritin technology (Carter & Li, 2003; Li, et al., 2006) involves the creation of novel functionalities from an existing naturally occurring and ubiquitous ferritin nanoparticle involved in iron storage. Ferritin is comprised of a small 17 kd protein which self assembles into a spherical 24 unit capsid with a hollow core (FIG. 1). The fortuitous positioning of the N- and C-termini of each subunit on the outer and inner core of the capsid respectively, allows for the engineering of novel materials by standard genetic engineering practices. The surface exposed positions of these termini provide a scaffold to genetically engineer an immense variety of novel nanomaterials with therapeutic, diagnostic and electronic applications. For example, in potential oncology applications, the genetically engineered ferritin containers can be used to house therapeutic drugs and diagnostics, while surface modifications can be used to direct the capsid for highly specific drug delivery or for the creation of new vaccines.
As part of the inventor's early foundational work with ferritin fusion proteins, applications were demonstrated in several areas, including vaccine development (e.g., HIV) and nanomaterial synthesis (e.g., silver single crystals condensed in the core with novel metal binding peptide fusion), as well as demonstration of solution plasmon resonance (Kramer, et. al, 2004). The present inventor observed, and now many others have confirmed, the ease, rapidity and relatively inexpensive process with which these fusion products can be made using standard recombinant techniques and a full spectrum of industry standard prokaryotic and eukaryotic expression systems. Ferritins with novel functionalities can be made and examined in as little as 10 days and modern high-throughput methods allow for the potential production of dozens of these genetic constructs in parallel.
In the vaccine application alone, there are broad and far reaching implications for the successful outcome in a variety of deadly diseases, many which are endemic throughout the world, including influenza and the promise of the long awaited HIV vaccine. To this end, NIH researchers have contributed an additional beautiful example of the effectiveness of this technology in animals against H1N1 influenza (Kanekiyo et al, 2013). In an issue of Science Magazine, the prior ferritin technology has been heralded as the answer to the long awaited universal flu vaccine (“Once-in-a-Lifetime Flu Shot?” Science Vol 341: pg. 1171, September, 2013) (FIG. 2). Clearly, the great potential of the ferritin non-viral nanoparticle platform (Carter & Li, 2003) has been validated independently by a number of research groups around the world.
The protein known as non-structural protein 10 or NSP10 is a viral regulatory protein found in at least the Group I, II and III coronaviruses. The three-dimensional atomic structure of NSP10 from the SARS coronavirus was determined by Su, et al., (2006) (FIG. 3). See also Joseph et al. (2006). This is an approximate 17 Kd MW viral gene regulatory/replicase-inhibitor protein that binds to the host cell 40S ribosomal unit and inhibits translation of host proteins. By suppressing host cell expression, NSP10 facilitates the production of its own viral gene expression.
Structurally, NSP10 is categorized as a zinc finger protein and can be further described as a two subdomain structure with one n-terminal helical subdomain (subdomain I) and one c terminal small beta sheet subdomain (subdomain II). NSP10 normally self-assembles into a spherical dodecamer having trigonal 32 point symmetry with an outer diameter of approximately 84 Å and an inner hollow hydrophobic chamber of 36 Å in diameter (FIGS. 3 & 4) (Su, et al., 2006; PDB identifier: 2G9T, sequence identifier P0C6U8). Subdomains I self-associate to form a trimer interaction at the four capsid n-terminal three-fold axes and subdomains II self-associate as trimeric units on the four c-terminal three-fold axes. One zinc binding site occurs at the interface between the two subdomains, and the three other zinc sites are located within subdomain II near the c-terminus.
NSP10 remains a unique topological representative of a structurally distinct assembling family of proteins, despite almost a decade since its first discovery. There have only been implied sequence homologies with other proteins, such as the HIT-type zinc finger proteins identified through sequence homology by Su, et al. (2006) (FIG. 5) which are also believed to be involved in gene regulation. Given the identified gene regulatory role of this protein, it would be understood that other topologically similar proteins exist, and thus by referring to NSP10, this includes other proteins that have the same physical folds, dimensions or properties, and are NSP10-like (or “NSPL”). In addition, NSP10 as used in the present application refers to other proteins having the same properties of folding and self-assembly as the NSP10 protein. Other proteins usable in the present invention will have sequence homology with the NSP10 protein in varying degrees, such as any level of 45% sequence homology of higher, e.g., 50% homology, 55% homology, 60% homology, 65% homology, 70% homology, 75% homology, 80% homology, 85% homology, 90% homology, 95% homology, or higher. The NSP10 proteins of the invention will thus include those proteins that may not have at least 45% sequence homology, but which contain similar binding regions and bonding patterns such that the self-assembly of the molecule forms the same pattern as the NSP10 fusion protein.
It is thus possible to develop alternate amino acid sequences of NSP10 proteins and accomplish the same objectives of the invention described herein. For example, it would be understood that amino acid substitutions to increase stability, remove zinc binding or change the amino acids exposed in the interior, would be considered within the scope of the invention as set forth below provided that the NSP10 self-assembles as indicated above. As a detailed example, the NSP10 proteins of this invention constitute a family of proteins that have important inter-subunit contacts which occur at the 2 folds of the capsid. Here, the main surface interaction is between two beta sheets running antiparallel (FIG. 6). If necessary or desirable, such features call for the improvement of capsid stability by replacing Met 44 on each protein by cysteine to potentially form a crosslinking disulfide bridge between two fold related dimers. The intermolecular distance between these two residues is approximately 6.2 Å. In the same line of reasoning, one may also potentially substitute a cysteine at or near Valine 42 which could potentially form a disulfide with Cysteine 46 of the adjacent molecule. In any case, it would be readily possible without undo experimentation to create a nanoparticle with increased stability by cross linking the protein in this manner, whether at the two-fold, or elsewhere on the molecule.
Moreover, very recent advances in protein structure prediction and engineering design have made it possible to design new capsid proteins having no sequence homology with existing proteins, but creating the same oligomeric assembly, whether as an individual protein or as a two-component system (Bale et al., 2016). Such engineered proteins with the same similar topological features and the advantageous disposition of the n and/or c-termini for the fusion of proteins or peptides, would be considered within the scope of the invention as set forth below.
Although structurally unique (for example, there are no similarities in the three-dimensional topology of the individual NSP10 proteins with ferritin), they, like members of the ferritin family, are formed by the self-assembling monomeric units. There are no also amino acid sequence homologies between NSPL proteins and the ferritins. While the family of proteins as thus far described are zinc finger proteins, excess zinc is not required for the dodecahedron formation and assembly. Zinc, however, may be required for viral gene regulatory function (Su et al, 2006). The NSPL family of dodecahedrons are similar in size to the smaller dodecameric ferritin capsids induced by heat-shock (which also have nucleic acid binding ability, a property suggested to be protective association (stabilizing) functions). In dodecameric form (12 mer), they are approximately 84 Å in diameter vs. the 100 to 120 Å in diameter for normal 24 mer ferritins with 432 symmetry). In addition to the 12 mer assembly, there is a propensity for these to form dimers as discovered by the crystal structure. Surface electrostatic mapping reveals that the outer shell has definitive patches of positive charge (FIG. 4.) supportive of the proposed role in RNA regulatory processes (Su et al, 2006). Other distinctive features include a predominantly hydrophobic core structure (inner diameter of 36 Å) and examination of the structure space-filling model reveals a series of solvent assessable pores leading from the surface to the interior hollow core.
As previously described, ferritin nanoparticles possess an n-terminus that is located on the capsid surface making possible the display of peptide or protein fusions on the surface creating a VLP display. The types of surface display fusions are limited in this platform to the n-terminus, meaning that the fusion peptide or protein must be fused through the c-terminus of the fusion partner. This is referred to as an N to C terminal fusion requirement. The n-terminus is also located in close proximity to a capsid three-fold axis, making it possible to fuse and display natural oligomeric receptors which require three-fold symmetry. The c-termini of ferritin are located within the interior of the assembled capsid and very closely disposed around a four-fold axis. The c-termini are thus advantageously positioned to fuse peptides or proteins for interior modifications, such as changing the metal affinity and storage properties of ferritin (Kramer, et al, 2004). The c-termini, however are not advantageous in the surface display or the C to N terminal fusion required for a variety of other viral and receptor oligomeric structure requiring surface display and trimeric assembly. Examples of viral receptors that extend from the N to the C-terminus (Influenza, HIV, Ebola and coronaviruses) while other viruses have evolved receptor complexes which extend from the surface with a C to N terminus polarity (such as the reovirus and adenovirus families). Such viral receptors are the most important immunization targets and are key in eliciting neutralizing antibodies that prevent viral infection by blocking the viral receptor interaction and/or conformational requirements for subsequent membrane fusion. Information regarding the ferritin fusion proteins described above is shown in U.S. Pat. Nos. 7,097,841 and 7,608,268, both of these patents and their disclosures incorporated herein by reference.
When characterizing the NSP10 and the subsequent x-ray structure determination, Su et al. (2006) utilize a glutathione-S-transferase (GST) fusion protein for the affinity-based isolation, using a commercially available expression vector with the GST and a specialized protease cleavage site to remove the target protein from the GST. In this way the GST component remains bound to the column and the target protein is easily eluted with relatively high purity. In this manner, Su et al. obtained NSP10 material suitable for further characterization and crystallization. Consequently Su et al. did not evaluate the potential assembly of the GST fusion protein by itself.
As part of the work to evaluate the potential of the NSP10 family of proteins for capsid fusion applications it was necessary to examine the proteins with the fusion partners intact, something that was not demonstrated or suggested by Su et. al., nor since that time, anywhere in the literature. Here, we describe the utility of NSP10 proteins as identified above for a variety of nanoparticle fusion protein applications, and demonstrate for the first time the propensity for self-assembly and proper biological function of the fusion partners once assembled in the capsid form (Examples 1-6). By self-assembly is meant the ability of the protein when formed to fully or partially assemble into the established oligomeric structure including all folds, core regions, pores, and bonding. Self-assembly can also refer to the formation of an aggregate including the proteins of the fusion protein.
Unlike ferritin where the N and C-termini terminate on the exterior and interior of the capsid, respectively, the N and C-termini of NSP10 proteins both are perfectly disposed about the three-fold axes and both terminate on the capsid surface, thus providing a major advantage over prior art. This eliminates the polarity issue, previously described, which limits surface expression partners in ferritin. Most importantly, the termini of each are properly disposed about three-folds with inherent ideal spacing for the fusion of the receptor stems, either helical or fibrous in nature. This positioning creates an anchor point for nucleating the trimeric oligomeric structures of numerous and complex, viral and cellular receptors. The employment of three-fold symmetry created by a fusion partner is well known to catalyze or nucleate the correct folding of a trimeric component (Papanikolopoulou, et al., 2004). Accordingly, the NSP10 proteins of the present invention will be able to be used in the same applications as described above for ferritin, but with the advantages as discussed herein.
As such, these protein or peptide fusions can be used to advantageously display the native form of various viral receptors for a more natural, improved antigen display or to guide the nanoparticle to a therapeutic target. Numerous virus families utilize the three-fold display of stems and receptors, these include the viruses of HIV, Ebola, influenza, coronaviruses, like SARS, MERS, and many others, some of which, like the orbiviruses, do not have an integral stem section, yet still utilize three-fold symmetry of the receptor/host recognition. This receptor display application of the NSP10 agents of the present invention agents can extend beyond viruses into cell tropism of many other infectious diseases and applications, including the targeted delivery of small molecule or protein therapeutic agents to cancerous cells or infectious agents, such as mycobacterial tuberculosis, and parasites, such as malaria. Clearly the scope of the possible applications of the novel NSP10 technology of the present invention is very broad and in addition to the applications described above, including those for the ferritin fusion protein, includes, for example, cell sorting, imaging, material science, vaccines, biosensors, diagnostics, and therapeutics, as described further below.
In this case, the assembled nanoparticle creates two unique sets of four identical three-fold related peptide terminal sequences, namely one set which terminates at the c-terminus and the other set which terminates at the n-terminus where the trimeric sets are each oriented in independent tetrahedral spatial configurations. As a conceptual and visual aide, since the NSP10 proteins of the invention have the same symmetry as a trigonal pyramid, each apex of the pyramid could be thought of as one terminal axis at the three-fold such as the three n-termini, while the c-termini three-folds can be represented by the center of each face of the pyramid (red or blue, see FIG. 7). A further graphic illustrates the final assembly of a c-terminal fusion with a viral stem and receptor (FIG. 8).
Further, an additional set of 4 stem fusions can be constructed on the same particle with the remaining 4 sets of trimeric N termini. FIG. 8 also illustrates the lack of steric spatial restrictions for these fusions, including the large GST fusion tags used for affinity chromatography.